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Peter Morgan-Warren, Zubair Ahmed, Martin Berry, Robert A H Scott, Ann Logan; Pharmacological inhibition of GSK3β enables RGC neuroprotection and promotes neurite growth in cultured retinal cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4969. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
There is emerging evidence that the activity of the intracellular kinase glycogen synthase kinase 3 beta (GSK3β) is implicated in promoting axon regeneration after injury. We investigated the effects of pharmacological GSK3β inhibition on the survival of retinal ganglion cells (RGC) and RGC neurite outgrowth in culture.<br />
Retinal cultures were prepared from uninjured adult rats, seeded at 125000 retinal cells/well and treated with the aminopyrimidine derivative Chir99021, a highly potent pharmacological inhibitor of GSK3β, at concentrations of 30nM, 300nM, 3µM, 30µM and 300µM, or culture medium control. After incubation for 3 days, RGC were immuno-labelled for βIII-tubulin for analysis of survival and neurite outgrowth, and GSK3β and phosphorylated GSK3β (pGSK3β) antibodies to study cellular expression in RGC.<br />
Immunoreactive GSK3β was detected in 98.3±1.0% (mean ± SEM) RGC in control wells, and this frequency was not significantly altered after Chir99021 treatment at any test concentration. By contrast, immunoreactive pGSK3β was present in only a minority of RGC in control wells (16.4±5.3%) and these numbers were also not significantly altered after Chir99021 treatment at any test concentration. Compared to the number of surviving RGC in untreated control retinal cultures (316±22 RGC/well), treatment with Chir99021 was significantly RGC neuroprotective at concentrations of 300nM (512±52 RGC/well, p<0.05) and 3µM (544±79 RGC/well, p<0.01), but was RGC toxic at higher concentrations. GSK3β inhibition significantly increased the proportion of neurite-bearing RGC from 10.7±1.5% to 18.9±1.9% (p<0.05) and 18.1±2.1% (p<0.05) with 30nM and 300nM Chir99021, respectively, although this treatment had no significant effect on the length of neurites.<br />
Inhibition of GSK3β activity in adult rat retinal cultures with the potent and specific pharmacological antagonist Chir99021 does not alter levels of either immunoreactive GSK3β or pGSK3β in RGC. However, Chir99021 is RGC neuroprotective and promotes increased RGC neuritogenesis, with different optimal concentrations for survival and neurite growth, although this treatment does not increase the length of RGC neurites. These results suggest that GSK3β may be a potential target for modulation in the development of future novel RGC neuroprotective and axon regenerative therapies.<br />
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